Technical Abstract:
Organoleptic qualities of processed and formulated foods are primary determinates of consumer acceptability. Stabilization of milk proteins in acid dairy drinks, gelation in jams and jellies, and texture-firming ionic interactions in fruits and vegetables are product qualities mediated by structural properties of the polysaccharide pectin. The goal of this project is to create the knowledge and skill required to develop an enzyme-based technology for precise engineering of pectin’s nanostructure. We hypothesize pectin nanostructure can be enzymatically engineered with pectin methylesterase (PME) to design and tailor novel functionality for improved consumer properties and fulfill well defined product qualities not available by conventional formulation or processing. The objectives are to: 1) purify and characterize PME from an industrial enzyme preparation obtained from papaya (Carica papaya) and correlate identity to endogenous fruit PME(s), 2) prepare a demethylated pectin series from a highly methylated model homogalacturonan (HG) substrate with the well-defined PME, 3) characterize the nanostructure of PME-modified pectin, 4) model the enzyme’s mode of action and degree of processivity, and 5) determine rheological properties of the modified HG and correlate them to the introduced nanostructural modifications. Results to date include: A single,protease-stable PME present in the preparation was purified to homogeneity and its biochemical and structural properties were characterized. Several demethylation series were produced using a highly methylated model HG and by varying reaction pH, enzyme-substrate ratios and following a limited initial demethylesterification by a fungal PME. Nanostructural properties (average demethylated block size, number of demethylated blocks per molecule and enzyme degree of processivity) of PME-modified HG were characterized by HPAEC and the nanostructural properties were correlated to rheological properties.
Enzyme mode of action was modeled. This will enable predictive models to be constructed for producing pectin-based formulating agents having optimal and predictable physical properties.